This proposal aims to utilize the robust replication of an RNA virus to study gene function in disease vector mosquitoes using both forward and reverse genetic approaches. In the first aim, a portion of an RNA viral genome encoding the viral replicase will be expressed as a transgene from the mosquito genome in a tissue-specific manner, with a native mosquito gene sequence incorporated into the virus genome. As viral replication proceeds, RNA interference should be induced to both the viral RNA and the native mosquito gene. We expect that because of the viral replicase-mediated amplification step, linear variability in nuclear transcription rates, which affects the direct transcription of inverted repeat sequences and can be caused by transgene insertion sites, nutritional status or other factors, should be eliminated or substantially reduced. This should increase both the strength and the efficiency of RNAi-based reverse genetic experiments in mosquitoes as well as simplify the interpretation of RNAi knockout phenotypes. In the second aim, we seek to develop a forward genetic screen for mosquito genes involved in the RNA interference pathway. While the most obvious RNAi genes have been characterized, there are likely many critical components which remain unknown. A library-based recombinant virus system is proposed which will support virus replication if and only if a particular recombinant virus contains a portion of a gene involved in RNAi. Public Health Relevance. The mosquitoes Aedes aegypti and Anopheles gambiae are responsible for an enormous disease burden including dengue hemorrhagic fever and malaria. Developing methods to more precisely, more efficiently and more robustly interrupt mosquito gene function is critical to understanding the biology of these vectors, as well as understanding mechanisms of pathogen transmission. Understanding the genes involved in innate anti-viral immunity will allow better understanding of vector competence and modeling of transmission potential for introduced agents, either natural or through bioterrorism. [unreadable] [unreadable] [unreadable]